Efficient embryonic homozygous gene conversion via RAD51-enhanced interhomolog repair.

Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion.

WD repeat domain 48 promotes hepatocellular carcinoma progression by stabilizing c-Myc.

The role of protein members containing the WD40 repeat domain in many diseases, including cancer, is well documented. However, the role of WD repeat domain 48 (WDR48) in hepatocellular carcinoma (HCC) and its molecular basis remain to be further investigated. In the present study, we report that WDR48 is downregulated in clinical HCC samples and evaluate the relationship between its expression and clinical features of HCC. In vitro experiments showed that WDR48 positively regulated the proliferation, invasion and metastasis of HCC cells and in vivo experiments showed that downregulation of WDR48 significantly inhibited the tumorigenicity of HCC cells. Mechanistically, WDR48 binds to the proto-oncogene transcriptional regulator c-Myc and stabilizes c-Myc expression by mediating its deubiquitination, thereby enhancing cell proliferation and EMT signalling. Our study demonstrates the oncogenic role of WDR48 and suggests that WDR48 can be an important target in HCC.

The WD40-repeat protein WDR-48 promotes the stability of the deubiquitinating enzyme USP-46 by inhibiting its ubiquitination and degradation.

Ubiquitination is a reversible post-translational modification that has emerged as a critical regulator of synapse development and function. However, the mechanisms that regulate the deubiquitinating enzymes (DUBs) responsible for the removal of ubiquitin from target proteins are poorly understood. We have previously shown that the DUB ubiquitin-specific protease 46 (USP-46) removes ubiquitin from the glutamate receptor GLR-1 and regulates its trafficking and degradation in Caenorhabditis elegans We found that the WD40-repeat proteins WDR-20 and WDR-48 bind and stimulate the catalytic activity of USP-46. Here, we identified another mechanism by which WDR-48 regulates USP-46. We found that increased expression of WDR-48, but not WDR-20, promotes USP-46 abundance in mammalian cells in culture and in C. elegans neurons in vivo Inhibition of the proteasome increased USP-46 abundance, and this effect was nonadditive with increased WDR-48 expression. We found that USP-46 is ubiquitinated and that expression of WDR-48 reduces the levels of ubiquitin-USP-46 conjugates and increases the t1/2 of USP-46. A point-mutated WDR-48 variant that disrupts binding to USP-46 was unable to promote USP-46 abundance in vivo Finally, siRNA-mediated knockdown of wdr48 destabilizes USP46 in mammalian cells. Together, these results support a model in which WDR-48 binds and stabilizes USP-46 protein levels by preventing the ubiquitination and degradation of USP-46 in the proteasome. Given that a large number of USPs interact with WDR proteins, we propose that stabilization of DUBs by their interacting WDR proteins may be a conserved and widely used mechanism that controls DUB availability and function.

WD repeat protein WDR48 in complex with deubiquitinase USP12 suppresses Akt-dependent cell survival signaling by stabilizing PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1).

PHLPP1 (PH domain leucine-rich repeat protein phosphatase 1) is a protein-serine/threonine phosphatase and a negative regulator of the PI3-kinase/Akt pathway. Although its function as a suppressor of tumor cell growth has been established, the mechanism of its regulation is not completely understood. In this study, by utilizing the tandem affinity purification approach we have identified WDR48 and USP12 as novel PHLPP1-associated proteins. The WDR48·USP12 complex deubiquitinates PHLPP1 and thereby enhances its protein stability. Similar to PHLPP1 function, WDR48 and USP12 negatively regulate Akt activation and thus promote cellular apoptosis. Functionally, we show that WDR48 and USP12 suppress proliferation of tumor cells. Importantly, we found a WDR48 somatic mutation (L580F) that is defective in stabilizing PHLPP1 in colorectal cancers, supporting a WDR48 role in tumor suppression. Together, our results reveal WDR48 and USP12 as novel PHLPP1 regulators and potential suppressors of tumor cell survival.

USP1-WDR48 deubiquitinase complex enhances TGF-ß induced epithelial-mesenchymal transition of TNBC cells via stabilizing TAK1.

Triple-negative breast cancer (TNBC) is the most aggressive histological subtype of breast cancer and is characterized by poor outcomes and a lack of specific-targeted therapies. Transforming growth factor-ß (TGF-ß) acts as the key cytokine in the epithelial-mesenchymal transition (EMT) and the metastasis of TNBC. However, the regulatory mechanisms of the TGF-ß signaling pathway remain largely unknown. In this study, we identified that the USP1/WDR48 complex could effectively enhance TGF-ß-mediated EMT and migration of TNBC cells. Furthermore, lower phosphorylation of Smad2/3, Erk, Jnk, and p38 was noted on the suppression of the expression of endogenous USP1 or WDR48. Moreover, the USP1-WDR48 complex was found to downregulate the polyubiquitination of TAK1 and mediate its in vitro stability. Therefore, our findings have shed a light on the novel role of the USP1/WDR48 complex in promoting TGF-ß-induced EMT and migration in TNBC via in vitro stabilization of TAK1.

The Role of the Complex USP1/WDR48 in Differentiation and Proliferation Processes in Cancer Stem Cells.

BACKGROUND: Recently, some studies identified the Basic-Helix-Loop-Helix (bHLH) transcription factor as a significant regulator for the evolution of neoplasms. The binding between bHLH proteins and DNA is restricted by heterodimerization with Inhibitors of DNA binding (ID). IDs prevent cellular differentiation, promote growth and sustain tumor development. The wide presence of stem cells in cancers suggests that genes ID are essential to cancer stem cells (CSC) progress. The enzyme Ubiquitin-specific protease 1 (USP1) is reported to deubiquitinate and stabilize IDs. Considering the action of the proteins ID, USP1 contributes to prevent differentiation mediated by bHLH and, consequently, keep CSC original characteristics. USP1 has its activity potentiated when bound to protein WD repeat-containing protein (WDR48). OBJECTIVE: To identify the influence of the complex USP1/WDR48 during the CSC tumorigenesis process, and whether this complex is a possible therapeutic target. METHODS: A literature search regarding the role of the complex USP1/WDR48 in inhibiting differentiation and increasing proliferation of CSC was performed, and possible selective molecule inhibitors of these deubiquitinase proteins were investigated. RESULTS: There is evidence that USP1/WDR48 complex promotes stem cell conservation and regulation of DNA damage repair. For this reason, inhibitors as Pimozide, GW7647, C527, SJB2-043, ML323 have been studied to inhibit USPs in cases of treatment intervention. CONCLUSION: It is consolidated in the literature the role of USP1/WDR48 during tumorigenesis. However, these studies are not enough to completely clarify the process; but certainly, the researchers are converging towards a promising direction to provide a new treatment option for cancer.

Function of the Deubiquitinating Enzyme USP46 in the Nervous System and Its Regulation by WD40-Repeat Proteins.

Posttranslational modification of proteins by ubiquitin regulates synapse development and synaptic transmission. Much progress has been made investigating the role of ubiquitin ligases at the synapse, however very little is known about the deubiquitinating enzymes (DUBs) which remove ubiquitin from target proteins. Although there are far fewer DUBs than ubiquitin ligases encoded by the human genome, it is becoming clear that DUBs have very specific physiological functions, suggesting that DUB activity is tightly regulated in vivo. Many DUBs function as part of larger protein complexes, and multiple regulatory mechanisms exist to control the expression, localization and catalytic activity of DUBs. In this review article, we focus on the role of the DUB USP46 in the nervous system, and illustrate potential mechanisms of regulating DUBs by describing how USP46 is regulated by two WD40-repeat (WDR) proteins, WDR48/UAF1 and WDR20, based on recent structural studies and genetic analyses in vivo.